| In recent years, marine oil spills occur frequently due to kinds of natural and anthropogenic activities. These accidents cause serious harmful effect to marine environment and our sustainable development of society directly or indirectly. Currently, there are many strategies to dispose the spilled oil. Among these strategies, bioremediation method has the characteristics of green, efficient and cheap and becomes the best potential method to clean up the spilled oil. However, lots of questions still need to be solved during application of bioremediation, such as keeping high biodegrading activity of exogenous bacteria and enhance environmental adaptability of biodegrading bacteria. So, facing the complicated situation, immobilization technology would be the best methods to deal with such problems. In this work, marine oil-degrading bacterium Marinobacter sp PY97 S was used as the target strains. Microspheres were prepared by immobilizing the target bacteria and bioremediation were applied to clean up the spilled oil on coastline. What we get were as followings:Polyvinyl alcohol(PVA), sodium alginate and activated carbon were used as bio-carriers to immobilize the marine oil-degrading bacteria. The optimum immobilization method was estimated by analyzing the mechanical properties, mass transfer characters, grain size, and bacterial contents of the immobilized microsphere. The results showed that the bacterial immobilized microsphere could be easily and successfully manufactured by embedding with the hydro-gel composed of 40-60 g/L PVA, 20-30 g/L sodium alginate and 5 g/L activated carbon. Meanwhile, the developed network structure interlaced within the microsphere. This complicated structure provides enough space for bacteria to attach and growth.Based on the above results, three kinds of immobilized microspheres were made with different material components. Gravimetric method and GC-MS were used to determine oil degradation effect. The results showed that all of the immobilized microspheres degraded more than 30% of crude oil in 12 d. Compared with free bacteria(FB), the degradation rate of S8 immobilized microsphere(composed of 60 g/L PVA, 20 g/L sodium alginate and 5 g/L activated carbon) increased by 7%, which showing the best oil biodegradability. Through biodegrading spectrum analysis by GC/MS, the removal rates of n-alkanes and polycylclic aromatic hydrocarbon(PAHs) increased significantly. The degradation rates of these two pollutants were 30% and 45% in 12 d, respectively. Beside, the fluorescent quantitative PCR showed that the bacteria increased steadily within the microsphere during the degrading process.In this study, treatments of natural storage, 4 oC storage and freeze drying storage were studied to preserve the immobilized microsphere. The bacteria content and its oil-degrading ability were evaluated. The results showed that the bacteria content and oil-degradation ability remained stable in 30 d in different samples. After then, the parameters declined gradually in the former two samples. On the contrary, the bacteria could maintain superior bioactivity in the freeze drying microsphere, accompanied by degraded more than 40% crude oil. Thus, it would be the better way to store the immobilized microsphere by freeze drying method.In this part, the experiment was carried out for 129 d after the oil spill accident of Huangdao pipeline explosion in 2013. The residual oil between the sediments of bioremediation and non-bioremediation was measured by gravimetric method and GC-MS. What we learned that the residual oil varied in a large range, showing up and down irregularly in the non-bioremediation area, while it decreased continuously in the bioremediation area. Considering the oil degradation rate, the immobilized oil degrading bacteria accelerated the removal of spilled oil by enhancing 53.7% compared with the control area in 30 d. What is more, after bioremediation of 129 d, the spilled oil was degraded 67% in the bioremediation area, while it just 46% in the control area. Meanwhile, the GC-MS results showed that the content of the main component of n-alkanes and PAHs had obviously difference between the bioremediation area and non-bioremediation area.The microbial biodiversity in the microsphere was investigated by MiSeq sequencing method during the biodegradation period. The results showed that the microbial biodiversity were increasing in natural environment. Proteobacteria and Bacteroides play an important role during the bioremediation experiment, as they were the key members within the microsphere. Community structure analysis showed that numbers and genus of different functional bacteria cooperated in the microspheres. What is more, analysis of microbial community inside the microsphere showed that the exogenous bacterium Marinobacter could steadily reside in the microsphere except a small amount of loss. This indicated that immobilized bacteria could adapt to adverse environment and keep long term oil degradation ability.In conclusion, the prepared microspheres of immobilized bacterial had good physical properties and oil degradation activity. Through in situ bioremediation application of spilled oil on coastline, it indicated that the immobilized bacterium Marinobacter sp PY97 S could accelerate and enhance oil-biodegrading ability, promoting the improvements of contaminated environment. |
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